Analysis of p73 expression pattern in acute myeloid leukemias: lack of DN-p73 expression is a frequent feature of acute promyelocytic leukemia MG Rizzo 1 , E Giombini 1 , D Diverio 2 , M Vignetti 2 , A Sacchi 1 , U Testa 3 , F Lo-Coco 4 and G Blandino 1 1 Department of Experimental Oncology, Regina Elena Cancer Institute, Rome, Italy; 2 Department of Cellular Biotechnologies and Hematology, University ‘La Sapienza’, Rome, Italy; 3 Laboratory of Hematology and Oncology, Istituto Superiore di Sanita `, Rome, Italy; and 4 Department of Biopathology, University ‘Tor Vergata’, Rome, Italy p73, the homologue of p53, is a nuclear protein whose ectopic expression, in p53 þ / þ and p53/ cells, recapitulates the most well-characterized p53 effects, such as growth arrest, apoptosis and differentiation. Unlike p53, which is mutated in half of human cancers, p73 is rarely mutated. However, altered expression of the p73 gene has been reported in neuroblasto- ma, lung cancer, prostate cancer and renal cell carcinoma. To investigate the potential involvement of p73 in acute myeloid leukemias (AMLs), we analyzed 71 samples from AML patients for the expression pattern of N-terminal transactivation-p73a (TA-p73a), its spliced isoforms and N-terminal-deleted-p73 transcripts (DN-p73). We detected p73 gene expression in AML irrespective of FAB (French–American–British) subtypes. Notably, the analysis of DN-p73 expression, which has been reported to inactivate both p53 and p73 antitumor effects, revealed a rather peculiar pattern. In fact, DN-p73 transcript and protein were detectable in 27/28 (96.4%) cases of M0, M1, M2, M4, M5 and M6 AML and in 13/41 (31.7%) cases of PML-RARa- positive M3 AML (Po0.01). Thus, the distinct gene expression profile of p73 further supports the notion that acute promye- locytic leukemia is a biologically different subset of AML. Leukemia (2004) 18, 1804–1809. doi:10.1038/sj.leu.2403483 Published online 16 September 2004 Keywords: acute myeloid leukemia; acute promyelocytic leukemia; p73; isoform Introduction p73, the recently discovered p53 family member, is a nuclear protein that shares a remarkable homology, both at sequence and protein levels, with p53. 1 In common with p53, the p73 protein shows three key functional domains: (a) the N-terminal transactivation domain, which shares 29% homology with the N-terminal part of p53; (b) the sequence-specific DNA-binding domain, which shares 63% homology with the corresponding p53 domain and (c) the tetramerization domain, which shares 42% homology with the oligomerization domain of p53. 2 The three-dimensional structure of the C-terminal tail of p73 has recently been solved by nuclear magnetic resonance spectro- scopy. 3 It consists of a five-helix bundle characterized by a marked similarity to the structure of sterile a motif (SAM). These domains are known to be protein–protein interaction modules present in several cytoplasmic signaling proteins and in transcription factors. Unlike p53, the p73 gene encodes several polypeptides. Two p73 polypeptides were originally identified. 1 The longer one, named p73a, comprises 636 amino acids. The shorter one, named p73b, derives from an alternative splicing of exon 13. Additional p73 isoforms that arise from diverse alternative splicings at the C-terminus have recently been identified 4–8 (Figure 1). Amino-terminally truncated isoforms (DN-p73) that lack the transactivation domain and exert dominant-negative function towards p53, p73 and p63 activity have been described 9–13 (Figure 1). These latter isoforms take origin from a cryptic promoter located in the third intron of the p73 gene 9 (Figure 1). Ectopic expression of p73 isoforms in both p53 þ / þ and p53/ recapitulates the well-characterized p53 antitumoral effects, such as growth arrest, apoptosis and differentiation. 14–16 These effects mainly occur through the activation of common and distinct target genes compared to those recruited by wild- type p53. 17 Unlike p53, which represents the most frequently mutated gene in human cancers, p73 is rarely mutated. 1,18–21 Despite its localization in a genomic region frequently altered in neuroblastoma and other cancers, there is still scarce evidence supporting a role of p73 in the pathogenesis of any specific human tumor. As to hematologic neoplasms, no mutations of this gene have been detected in a recent survey including most common myeloproliferative and lymphoproliferative malignancies. 21 p73-deficient mice exhibit severe defects, including hydro- cephalus, hypocampal dysgenesis, chronic infections and inflammation and abnormalities in the pheromone sensory pathway. 9 It has also been reported that p73 mRNA is upregulated during differentiation of muscle, neuronal and hematopoietic cells. Ectopic expression of p73a promotes neuronal and hematopoietic differentiation. 16,22,23 Here, we have investigated the expression pattern of TAp73a, its spliced isoforms and DN-p73 in diagnostic samples derived from patients with acute myelogeneous leukemia (AML), representative of all major morphologic and genetic subsets. We detected p73 expression in all AML types but significantly different DN-p73 expression patterns in APL as opposed to other AMLs. Materials and methods Patient samples and RNA preparation Leukemia samples were obtained from peripheral blood or bone marrow specimens collected at diagnosis from 71 AML patients. All patients were diagnosed and treated at the Department of Human Biotechnology and Hematology of the University ‘La Sapienza’ of Rome. Informed consent was obtained from the patients or their parents. The series was representative of the main morphologic subtypes according to the FAB classification system 24 and included the following forms: M0 (five samples), M1 (five samples), M2 (five samples), M3 (41 samples), M4 (five samples), M5 (five samples) and M6 (five samples). With concern to genetic characterization, all M3 cases were featured by the presence of the t(15;17) translocation and/or the PML/ Received 29 October 2003; accepted 16 July 2004; Published online 16 September 2004 Correspondence: Dr MG Rizzo, Department of Experimental Oncology, Regina Elena Cancer Institute, Via delle Messi d’Oro 156, Rome 00158, Italy; Fax: þ 39 06 4180 526; E-mail: rizzo@ifo.it Leukemia (2004) 18, 1804–1809 & 2004 Nature Publishing Group All rights reserved 0887-6924/04 $30.00 www.nature.com/leu